Biomedical subjects
Publications and source records attributed to A M Lever.
Candida endocarditis of native valves is difficult to diagnose and treat. The majority of cases require valve replacement and long term amphotericin treatment. We describe a case of Candida endocarditis which was successfully managed with fluconazole without valve replacement. The patient has now been reviewed for 4.5 years from the time of diagnosis and 39 months since treatment was discontinued.
The genetic approach to HIV infection is still very young and a number of different stages in the viral life cycle are being studied as targets for gene therapy, using a wide variety of modalities for gene delivery. Several gene therapy protocols for AIDS have been approved and when this article is published these may be in progress. In this chapter the scientific background to these in vivo studies is reviewed.
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Recombinant vectors based on the type 1 human immunodeficiency virus (HIV-1) can be used to deliver genes into cells expressing the HIV receptor, CD4. We have used a transient RNA packaging system to compare the safety and efficacy of HIV-1 vector transduction by wild-type and replication-deficient helper viruses. Helper virus-free vector transfer was consistently achieved when the helper virus gag-pol and env genes were expressed from separate plasmids such that two recombination events were required to form an infectious genome. Other forms of attenuation, such as deletion of the 5' phi region, were inadequate to prevent helper virus transmission. Vector transduction by the wild-type and non-replicating helper viruses occurred with comparable efficiency except in instances where efficient vector RNA expression was dependent upon transactivating factors supplied by the helper virus. These data demonstrate the feasibility of safe gene transfer using HIV-1 vectors.
We have previously described a series of human immunodeficiency virus type 1-based vectors in which efficient RNA encapsidation appeared to correlate with the presence of a 1.1-kb env gene fragment encompassing the Rev-responsive element (RRE). In this report, we explore in detail the role of the RRE and flanking env sequences in vector expression and RNA encapsidation. The analysis of a new series of vectors containing deletions within the env fragment failed to identify a discrete packaging signal, although the loss of certain sequences reduced packaging efficiency three- to fourfold. Complete removal of the env fragment resulted in a 100-fold decrease in the vector transduction titer but did not abolish RNA encapsidation. We conclude that the RRE and 3' env sequences are not essential for human immunodeficiency virus type 1 vector encapsidation but may be important in vectors in which a heterologous gene has been placed adjacent to the 5' packaging signal, potentially disrupting its structure.
A stable secondary structure model is presented for the region 3' of the primer-binding site to 130 bases into the gag sequence of the prototype type D retrovirus Mason-Pfizer monkey virus. Using biochemical probing of RNA from this region in association with free energy minimization, we have identified a stem-loop structure in the region, which from other studies has been shown to be important for genomic RNA encapsidation. The structure involves a highly stable stem of five G-C pairs terminating in a heptaloop. Comparison of the Mason-Pfizer monkey virus structure with one predicted for squirrel monkey retrovirus demonstrates an identical stem and a common ACC motif in the loop. Free energy studies of the secondary structure of the 5' regions of eight other retroviruses predict stem loops which have similar GAYC motifs. We believe this may represent a common structural and sequence motif which among other functions may be involved in genomic RNA packaging in these viruses.
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The location of HTLV-I (human T-cell leukemia virus type 1) proviral sequences in the genome of infected human cells was explored by hybridization of a viral probe with compositional fractions of host-cell DNAs. In the twelve cases examined, HTLV-I sequences were absent from the GC-poorest 40% of the host genome (namely, from isochores that are below 39% GC). Transcriptionally inactive proviral sequences were localized in GC-poor isochores (comprised between 39% and 42-44% GC) of the human genome, which are characterized by a constant and low gene concentration. In contrast, transcriptionally active proviral sequences were found in the GC-rich and very GC-rich isochores, which are gene rich, transcriptionally and recombinationally active, and endowed with an open chromatin structure. Since GC-rich isochores are present in R'-bands and very GC-rich isochores form T-bands, these results also provide information on the location of HTLV-I proviral sequences in human chromosomes. The results obtained with HTLV-I are in agreement with the non-random, compartmentalized integration of animal retroviral sequences that had been previously observed in other viral-host systems. They provide, however, much more detailed information on the regional location of proviral sequences in the host genome and on the correlation between their transcription and their location.
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Severe infections with Pseudomonas pseudomallei, the causative agent of melioidosis, is a common cause of community acquired septicaemia in South East Asia and parts of Northern Australia, but infection in travellers returning to the United Kingdom is extremely rare. We describe the case of a tourist who acquired melioidosis in Thailand. Despite intensive intravenous therapy with antibiotics to which the organism was sensitive, the patient's infection proved fatal. Ps. pseudomallei was isolated from blood cultures at a late stage in his illness by use of a commercial blood culture system that included an antibiotic removal device (Bac/TAlert, Organon-Technica).
OBJECTIVES: To investigate the presence of antibodies to HTLV and HIV retroviral antigens in the rheumatological diseases rheumatoid arthritis (RA), polymyositis/dermatomyositis (PM/DM), primary Sjögren's syndrome (pSS), and systemic lupus erythematosus (SLE), and to use polymerase chain reaction (PCR) to seek these exogenous retroviruses in proviral form in cellular DNA from these patients. METHODS: Thirty patients with active RA, 13 with PM, 14 with pSS and five with SLE were recruited and their sera tested for antibodies to HTLV-I in enzyme linked immunosorbent assay (ELISA) and Western blot analysis. Seropositivity to HIV-1 was also sought. DNA was extracted from peripheral blood lymphocytes, synovial tissue and muscle biopsies and tested by polymerase chain reaction using consensus primers for HTLV-I and HIV-1. RESULTS: In HTLV-I ELISA, nine rheumatological sera (4/30 RA, 3/13 PM/DM and 2/5 SLE patients) were considered positive; 14 from pSS patients and 30 from normal subjects were negative. In a control group which included osteoarthritis, Crohn's disease and bacterial endocarditis patients, only two of 80 proved positive in this system. Validation of these sera by Western blotting generally revealed weak reactivity against a variety of HTLV-I antigens. PCR of genomic DNA derived from patients' peripheral blood mononuclear cells did not reveal the presence of HTLV-I and HIV-1 target sequences. CONCLUSIONS: This study shows that PCR precludes HTLV-I and HIV-1 infection as causative agents in these rheumatological diseases although a minority of patients possess antibodies that are weakly cross-reactive with retroviral antigens.
cis elements required for the encapsidation of human immunodeficiency virus type 1 (HIV-1) RNA have been investigated by using a replication-competent helper virus to package a series of HIV-1-based vectors which had been stably transfected into human CD4 T-cell lines. A previously identified packaging signal in the 5' leader region was not sufficient for the encapsidation of small vectors containing heterologous genes. In contrast, vectors containing additional gag and env sequences were packaged with high efficiency and transduced into CD4-expressing target cells with titers exceeding 10(4) CFU/ml. The presence of gag sequences did not enhance vector packaging efficiency. A 1.1-kb env gene fragment encompassing the Rev-responsive element was absolutely required for the expression and encapsidation of vectors containing cis-acting repressive sequences and appeared also to contain an important packaging signal. Vectors as small as 2.6 kb were successfully packaged in this system. The presence of abundant, packageable vector RNA did not appear to interfere with encapsidation of the wild-type HIV-1 genome, suggesting that HIV-1 RNA packaging capacity is not saturated during acute infection.
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Interaction of cis-acting RNA sequences with nucleocapsid proteins is one of the critical events leading to retroviral genomic RNA packaging. We have derived a potentially stable secondary structure for the packaging signal region of human immunodeficiency virus strain IIIB, using a combination of biochemical analysis and computer modelling. This region encompasses the major splice donor (SD), which is found in a highly structured conserved stem-loop. Comparison with other published human immunodeficiency virus type 1 sequences shows almost absolute nucleotide conservation in base-paired regions required to maintain this structure. Presently and previously described packaging-defective mutants would disrupt the structure. The structure depends on base pairing between nucleotide sequences 5' of the major SD which are common to both genomic and subgenomic RNAs and sequences 3' of SD which are unique to the unspliced RNA. This may explain how in retroviruses such as Rous sarcoma virus, mutations in regions common to genomic and subgenomic RNA might prevent packaging of the unspliced mRNA by disrupting a signal structure which can exist only in the genomic RNA species.
This overview has attempted to cover the more common aspects of the clinical presentations of HIV infection and the differential diagnoses. These can only be generalizations as presentations differ dramatically from patient to patient. It is also important to be aware that in an individual the disease is continually evolving and the same complication may produce very different clinical manifestations at different times. It is vital to have a high index of suspicion of the occurrence of complications and to treat promptly. Delay may not only make successful treatment more difficult but it is conceivable that it may exacerbate the underlying immunodeficiency and hasten progression of disease.
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